Endocytosis and trafficking within the endocytosis pathway are known to modulate the activity of different signaling pathways. Epsins promote endocytosis and are postulated to target specific-proteins for regulated endocytosis. A functional link is presented between the Notch pathway and epsins. The Drosophila epsin liquid facets has been identified as an inhibitor of cardioblast development in a genetic screen for mutants that affect heart development. lqf inhibits cardioblast development and promotes the development of fusion-competent myoblasts, suggesting a model in which lqf acts on or in fusion-competent myoblasts to prevent their acquisition of the cardioblast fate. lqf and Notch exhibit essentially identical heart phenotypes, and lqf genetically interacts with the Notch pathway during multiple-Notch-dependent events in Drosophila. The link between the Notch pathway and epsin function is extended to C. elegans, where the C. elegans lqf ortholog acts in the signaling cell to promote the glp-1/Notch pathway activity during germline development. These results suggest that epsins play a specific, evolutionarily conserved role to promote Notch signaling during animal development and support the idea that they do so by targeting ligands of the Notch pathway for endocytosis (Tian, 2003).
Phenotypic studies of lqf embryos are consistent with the model that lqf acts in a subpopulation of fusion competant myoblasts (FCMs) to inhibit their acquisition of the cardioblast fate. How might lqf and Notch inhibit cardioblast development? In lateral and ventral regions of the mesoderm, Notch-mediated lateral inhibition helps select individual somatic muscle progenitor cells from clusters of equipotential cells. Cells in these clusters express lethal of scute (l'sc) and can adopt either the muscle progenitor or FCM fate. Cells that retain l'sc become progenitor cells while cells that lose l'sc expression become FCMs. In these clusters, Notch inhibits l'sc expression and the progenitor fate, thereby promoting the FCM fate. It is speculated that lqf and Notch may act similarly to regulate the cardioblast progenitor/FCM decision in the dorsal mesoderm with Notch functioning to inhibit tinman expression and the cardioblast progenitor fate and, in so doing, promoting the FCM fate. In this model, loss of lqf/Notch activity would lead to excess cardioblast progenitors at the expense of FCMs. Consistent with this, clusters of Tin-expressing cells in the dorsal mesoderm resolve to individual heart cells during the stages when lqf and Notch inhibit cardioblast development (Tian, 2003).
If lqf plays a general role in the promotion of Notch activity, why are defects observed only during heart development in lqf embryos? One explanation is that maternal lqf product masks earlier requirements for lqf during Notch-dependent events. Consistent with this, temperature shift experiments indicate lqf acts during late stage 12 to restrict cardioblast development. Nearly all well-characterized Notch-dependent events in the embryo occur before stage 12. Thus, the apparent specificity of the lqf phenotype for the heart may simply arise due to the late stage at which this Notch-dependent event occurs, combined with the masking effect of lqf maternal product. Unfortunately, it was not possible to assay embryos devoid of maternal and zygotic lqf function since lqf germline clones failed to produce eggs (Tian, 2003).
Regarding the function of C. elegans epsin during postembryonic development, upon Ce-epn-1 RNAi treatment, a weak glp-1 loss-of-function germline phenotype was observed, which was significantly enhanced in a glp-1 temperature-sensitive background at the permissive temperature. Strong lin-12 loss-of-function phenotypes were not observed in these experiments. The weak glp-1 loss-of-function and absence of lin-12 phenotypes in a wild-type background is very likely because the postembryonic feeding RNAi treatment only partially depletes Ce-epn-1 mRNA. The isolation and characterization of a null mutation will greatly facilitate uncovering all roles of Ce-epn-1 in C. elegans (Tian, 2003).
Since epsins appear to regulate endocytosis, and endocytosis regulates the activity of most signaling pathways (reviewed by Wendland, 2002), lqf might act broadly to regulate the output of many signaling pathways rather than acting specifically on the Notch pathway. However, existing data support a specific interaction between lqf and Notch activity. For example, no genetic interactions were observed between lqf and the EGF or FGF pathways during heart development, and lqf does not appear to interact with the dominant Egfr[ellipse] allele during eye development. Furthermore, lqf mutant clones in the Drosophila eye exhibit phenotypes consistent with the specific loss of Notch activity. Thus, lqf appears to display specific interaction with the Notch pathway. Although it is important to assay whether lqf alters the activity of other signaling pathways regulated by endocytosis, such as the TGFß, Wingless, and Hedgehog pathways, these data support a model in which Lqf plays a relatively specific role to target a component of the Notch pathway for endocytosis and in so doing promotes Notch signaling (Tian, 2003).
This work shows that lqf/epsins promote Notch pathway activity in Drosophila and C. elegans. Notably, epsins participate in Notch-mediated lateral inhibition signaling during bristle and perhaps heart development, as well as Notch-mediated inductive signaling in the C. elegans germline. These data argue that epsins are essential evolutionarily conserved components of the Notch pathway, potentially required for most if not all Notch-mediated processes (Tian, 2003).
Genetic studies have the potential to identify all components of a signaling process, however, they do not necessarily differentiate between the roles different genes play in a signaling process. A distinction can be made between core components of a signaling pathway -- those factors that actively take part in the signal transduction event -- and factors that set the stage for signal transduction but do not actively transmit the signal. For example, Notch, DSL ligands, presenillins and CSL effectors are core components of the Notch pathway as they actively transmit the signal -- DSL ligands bind Notch, induce the metalloprotease-mediated S2 cleavage followed by the presenilin dependent intramembrane (S3) cleavage of Notch that releases Notch[intra], which translocates to the nucleus and complexes with CSL-class proteins to activate Notch target genes. However, many other proteins set the stage for signaling by ensuring each core member of a pathway is present in the right location and correct form, such that signal transduction will occur given the proper stimulus. For example, presentation of a functional Notch receptor on the cell membrane appears to require S1-mediated cleavage of Notch by furin-type proteins. Although furins do not actively take part in the signaling event, furin activity and its requirement for presentation of Notch is a prerequisite for Notch signaling. Similarly, ras signaling requires Ras localization to the cell membrane and prenylation of Ras constitutively targets it to the cell membrane (Tian, 2003).
In support of lqf/epsins as core components of the Notch pathway, Delta endocytosis appears essential for Notch signaling and lqf appears essential for Delta endocytosis. Furthermore, epsins are thought to target ubiquitinated membrane proteins for regulated endocytosis via their ubiquitin-interacting motif (UIM) and ubiquitination of Delta appears necessary for Delta endocytosis and active Notch signaling. Thus, Lqf/epsins may act as part of a complex that specifically targets Delta for internalization after ubiquitination and as such be core members of the Notch pathway (Tian, 2003).
It remains possible, however, that lqf/epsin function is a prerequisite for Notch signaling. For example, some endocytic proteins function in protein transport in the secretory pathway and epsin1 family members could in principle enable transport of Delta to the membrane. In such a capacity, epsins would not be considered core components of the Notch pathway. Clearly, future experiments that test the requirement of specific domains of epsins, such as the UIM, for Notch signalling, as well as those that identify the protein complexes within which epsins act, should help elucidate the molecular basis through which lqf/epsins potentiate Notch signaling (Tian, 2003).
Fat facets is a deubiquitinating enzyme required in a cell communication pathway that limits to eight the number of photoreceptor cells in each facet of the Drososphila compound eye. Genetic data support a model whereby Faf removes ubiquitin, a polypeptide tag for protein degradation, from a specific ubiquitinated protein, thus preventing its degradation. Mutations in the liquid facets gene have been identified as dominant enhancers of the fat facets mutant eye phenotype. The liquid facets locus encodes epsin, a vertebrate protein associated with the clathrin endocytosis complex. Genetic experiments reveal that fat facets and liquid facets facilitate endocytosis and function in common cells to generate an inhibitory signal that prevents ectopic photoreceptor determination. The fat facets mutant phenotype is extraordinarily sensitive to the level of liquid facets expression. It is proposed that Liquid facets is a candidate for the critical substrate of Fat facets in the eye (Cadavid, 2000).
There are three key components of the endocytosis complex: (1) clathrin, which forms a cage structure engulfing the cell membrane; (2) AP-2, the core adaptor complex, which binds to clathrin and brings it to the cell surface, and (3) dynamin, a GTPase required for vesicle formation. Additional proteins associated with AP-2 have been identified, many of which contain protein-protein interaction domains called EH-domains and EH-domain-binding motifs. Epsin is an EH-domain-binding protein identified as a partner for Eps15, an EH-domain protein that also binds AP-2. The large number of AP-2-binding proteins identified suggests that many of them may have temporal and/or tissue-specific functions (Marsh and McMahon, 1999). The precise roles of Eps15 and epsin in endocytosis are unknown (Cadavid, 2000 and references therein).
The lqf gene itself is essential in Drosophila; in an otherwise wild-type background, lqf null mutants die as embryos. Clones of cells in the eye in which there is little or no lqf gene function have severely disrupted eye morphology, indicating that lqf is required also after embryogenesis for eye development. The mutant phenotypes associated with two weak lqf mutant alleles reveal specific roles for lqf in eye, wing and leg development (Cadavid, 2000).
The eye defects in homozygous adults for a weak allele of lqf resemble those in faf null mutants. As in faf mutants, the additional photoreceptors in lqf mutants arise from specific precursor cells (M-cells) present early during eye development. In contrast to lqf null mutants, faf null mutants are viable, have normal wings and legs and have less severe eye defects. Thus, lqf functions more broadly than faf, but both the lqf and faf genes are required during eye development in order to prevent the M-cells from becoming photoreceptors (Cadavid, 2000).
The faf mutant eye phenotype is unusually sensitive to a decrease in the dose of the lqf gene, suggesting strongly that the two genes function in a common pathway. Genetic interactions with endocytosis and Ub pathway mutants show that faf and lqf facilitate endocytosis and antagonize ubiquitination. In addition, although lqf is more broadly required than faf in the eye and elsewhere in the fly, weak lqf mutations reveal that like faf, lqf is required to prevent the misdetermination of M-cells as photoreceptors. Moreover, when expressed only in the rough positive cells surrounding the facet preclusters, both faf and lqf genes rescue completely to wild-type their respective M-cell misdetermination mutant phenotypes in the eye. Finally, given the relationship between Faf and its substrate protein, it would be expected that increasing the dose of the substrate should suppress the faf mutant phenotype. Slight overexpression of lqf completely obviates the need for faf in eye development. The simplest model consistent with all of this genetic data is that Lqf is the substrate of Faf. Other more complicated explanations are, of course, possible (Cadavid, 2000).
There is biochemical evidence that AF-6, a scaffolding protein thought to modulate cell-cell junctions in response to Ras activation may be an in vivo substrate of Fam, the mouse homolog of Faf; AF-6 and Fam bind each other in vitro and ubiquitinated AF-6 can be detected and deubiquitinated by Fam in cultured cells. Like lqf, the Drosophila Af6 homolog, canoe, is required pleiotropically for Drosophila eye development. In contrast to lqf mutations, however, canoe mutations do not act as strong dominant enhancers of the faf mutant eye phenotype. Given the striking genetic interactions between faf and lqf, it seems that canoe is unlikely to play a significant role in the essential faf pathway in the eye (Cadavid, 2000 and references therein).
While only one Faf/substrate interaction may be essential to normal eye development in Drosophila, Faf and Fam may have several substrates in vivo. Normally non-essential roles for faf later in eye development have been revealed in particular mutant backgrounds and Faf could have different substrates for its critical role in M-cell fate determination than in its redundant roles. Moreover, in addition to its essential role in eye development, faf is required maternally for cellularization of embryos and the critical maternal substrate of Faf is unknown. Because faf has mouse and human homologs, the modes of regulation by Faf are likely to be conserved. However, it is possible that the critical substrate(s) of Faf in Drosophila may differ from those in vertebrates (Cadavid, 2000 and references therein).
If Lqf is the substrate of Faf, then epsin levels, determined by the balance between its ubiquitination and deubiquitination, could regulate endocytosis. Mono-ubiquitination, however, has been shown previously to regulate endocytosis in two different ways. (1) Mono-ubiquitination of cell surface receptors can act as a signal for receptor endocytosis, which leads to lysosomal degradation. Here, the Ub moiety is somehow recognized by the endocytosis machinery; this process has nothing to do with the proteasome. (2) Eps15, an endocytosis complex component in mammalian cells, is mono-ubiquitinated in response to EGF receptor activation and Eps15 may require this modification to stimulate receptor endocytosis. In addition, Pan1p, a yeast protein similar to Eps15, is required for endocytosis in yeast. Although it is unknown whether Pan1p is mono-ubiquitinated in yeast, there is evidence that ubiquitination of an endocytic complex component is required for endocytosis in yeast; Rsp5p, a component of the ubiquitination machinery called a ubiquitin-ligase, may bind to Pan1p and is required generally for endocytosis in yeast, even for endocytosis of proteins with non-Ub endocytosis signals (Cadavid, 2000 and references therein).
Since Eps15 binds to epsin, could a mono-ubiquitinated Drosophila Eps15 homolog be the substrate of Faf? Two experimental results are inconsistent with this model. (1) It has been shown previously that the activity of Faf antagonizes proteolysis, not just ubiquitination; mutations in a gene encoding a proteasome subunit act as strong suppressors of the faf mutant eye phenotype. This result strongly suggests that Faf activity antagonizes proteolysis and thus that Faf deubiquitinates a protein containing a Ub chain targeting it for degradation, rather than a mono-ubiquitinated protein. (2) If mono-ubiquitination of Eps15 activates it, as the available data suggests, then deubiquitination of Eps15 by Faf would render Eps15 inactive and thus the function of Faf would antagonize endocytosis. The data presented here clearly indicate the opposite; mutations in endocytosis complex genes (particularly lqf and Clathrin heavy chain) act as strong dominant enhancers of faf, suggesting that the normal function of Faf is to facilitate endocytosis (Cadavid, 2000 and references therein).
Elevated levels of Lqf obviate the need for Faf, presumably by stimulating epsin-dependent endocytosis generally or stimulating endocytosis of a specific cell surface protein. How can the observation that Lqf and Faf function outside the M-cells to determine M-cell fate be reconciled with a role for Lqf in endocytosis? Endocytosis is known to modulate ligand/receptor interactions by a variety of mechanisms. One possibility is that M-cell fate is affected by a diffusible ligand that, like Wingless, travels via endocytosis through several cell distances. Alternatively, regulation of a membrane-bound receptor by endocytosis in cells adjacent to the M-cells could affect M-cell fate indirectly. For example, EGF receptor activity is downregulated by endocytosis following ligand binding. By contrast, activity of the Notch receptor may be up-regulated by endocytosis of activated receptors whose intracellular domains have been cleaved off prior to their translocation into the nucleus. Membrane-bound Notch receptors lacking their intracellular domains display dominant negative activity and endocytosis of cleaved Notch receptors may be required normally for precise modulation of Notch activity. Patterning of the photoreceptor preclusters in the developing eye may require that both Notch and the EGF receptor are activated in the rough-expressing cells surrounding the facet preclusters. Thus, Faf could regulate the activity of one or both of these receptors (Cadavid, 2000 and references therein).
Epsin is part of a protein complex that performs endocytosis in eukaryotes. Drosophila epsin, Liquid facets (Lqf), was identified because it is essential for patterning the eye and other imaginal disc derivatives. Previous work has provided only indirect evidence that Lqf is required for endocytosis in Drosophila. Epsins are modular and have an N-terminal ENTH (epsin N-terminal homology) domain that binds PIP2 at the cell membrane and four different classes of protein-protein interaction motifs. The current model for epsin function in higher eukaryotes is that epsin bridges the cell membrane, a transmembrane protein to be internalized, and the core endocytic complex. This study shows directly that Drosophila epsin (Lqf) is required for endocytosis. Specifically, Lqf is essential for internalization of the Delta (Dl) transmembrane ligand in the developing eye. Using this endocytic defect in lqf mutants, a transgene rescue assay has been developed and a structure/function analysis of Lqf has been performed. When Lqf is divided into two pieces, an ENTH domain and an ENTH-less protein, each part retains significant ability to function in Dl internalization and eye patterning. These results challenge the model for epsin function that requires an intact protein (Overstreet, 2003).
To test for endocytosis defects in lqf− mutants, the localization of the transmembrane receptor Dl was monitored in developing eyes. Dl normally undergoes endocytosis in the eye, and as the internalized protein is not degraded rapidly, internalized Dl can be detected in vesicles (Overstreet, 2003).
The Drosophila eye, composed of 800 identical 22-cell ommatidia, or facets, develops in the larval and pupal stages in a monolayer epithelium called the eye imaginal disc. Eye development occurs as a wave, where the morphogenetic furrow forms at the posterior of the disc, and moves anteriorly into the monolayer of undifferentiated cells. Rows of ommatidia assemble stepwise posterior to the furrow one or two cells at a time, starting with the eight photoreceptors (R1-R8) (Overstreet, 2003).
In wild-type, Dl is detected exclusively as intracellular dots within developing ommatidial clusters throughout the eye disc. In larval eye discs homozygous for lqfFDD9, a weak, viable mutant allele, Dl is detected mainly at the membranes of cells just posterior to the furrow. In clones of cells homozygous for lqfARI, a strong, lethal mutant allele, similar membrane localization of Dl is observed. It is concluded that lqf+ is required for Dl internalization (Overstreet, 2003).
All epsins have an amino-terminal ENTH domain that binds PIP2 at the cell membrane and three or four types of protein-protein interaction motifs, whose copy numbers vary among different epsins. The ubiquitin interaction motifs (UIMs) bind ubiquitin (Ub) noncovalently. There are also clathrin binding motifs (CBMs), DPW motifs that bind the core endocytic adaptor complex, AP-2, and NPF motifs that bind Eps15, another accessory factor (Overstreet, 2003).
A step toward understanding the role of Lqf in endocytosis is the identification of the modules of Lqf protein that are required. In yeast, there are straightforward assays for the function of the two epsins (Ent1 and Ent2). Structure/function analyses have demonstrated that the ENTH domain of Ent1 is necessary and sufficient to rescue the lethality of ent1Δent2Δ double mutants. Moreover, the ENTH domain and to a lesser extent the UIMs have been shown to be required for endocytosis. Because there are mechanistic differences between endocytosis in yeast and higher eukaryotes, the yeast epsins might function somewhat differently from vertebrate epsins and Drosophila Lqf. The major difference between these systems is that the AP-2 core adaptor complex in yeast has no known function in endocytosis, and, accordingly, the yeast epsins lack DPW motifs. As in yeast, structure/function analyses of epsins in vertebrate cell culture have pointed to the importance of the ENTH domain. These assays, however, rely on dominant-negative effects of mutant epsin proteins on endocytosis, and their interpretation is difficult (Overstreet, 2003).
Either the ENTH domain alone, or an ENTH-less Lqf protein, rescues the patterning and Dl endocytosis defects in lqfFDD9 homozygous eyes. Since experimental results in yeast and in vertebrates have emphasized the importance of the ENTH domain, the most remarkable result is that an ENTH-less Lqf protein can function. The simplest interpretation of the rescue results is that LqfΔENTH can function independently of the ENTH domain (Overstreet, 2003).
Transgenes that express Rat epsin1 or human epsin 2b in Drosophila with pRO, each as full-length proteins or without the ENTH domain, rescue the eye defects in lqfFDD9 homozygotes. Thus, there is unlikely to be a significant functional difference between the Drosophila and vertebrate epsins in the region C-terminal to the ENTH domain. In addition, the ENTH domains of Lqf and yeast epsin are functionally similar. It was shown previously that expression of the ENTH domain of Ent1, but not the complementary portion of the protein, restores viability to ent1Δent2Δ yeast. Similarly, expression of full-length Lqf or LqfENTH rescues ent1Δent2Δ lethality but LqfΔENTH expression does not (Overstreet, 2003).
Thus Drosophila epsin, Lqf, is essential for endocytosis of Dl in the developing eye. Moreover, the ENTH domain alone and an ENTH-less Lqf protein each retain significant function. The prevailing model in vertebrates is that epsin functions like a bridge, where the ENTH domain links the membrane to clathrin, a cell surface protein to be internalized, and to AP-2. Since this model requires an intact epsin protein, the results presented here suggest that the prevailing model cannot be the whole story. Moreover, the observation that either the ENTH domain or the remainder of the protein, which are functionally distinct, can be deleted without destroying Lqf function completely suggests that each fragment of Lqf may be partially redundant with another Drosophila endocytic protein. Candidates for the other endycotgic protein include the other ENTH domain protein in Drosophila, Epsin-2 and the Drosophila homolog of AP180, which, like the ENTH-less Lqf protein, binds clathrin and AP-2 (Overstreet, 2003).
Endocytosis modulates the Notch signaling pathway in both the signaling and receiving cells. One recent hypothesis is that endocytosis of the ligand Delta by the signaling cells is essential for Notch activation in the receiving cells. Evidence is presented in strong support of this model. In the developing Drosophila eye Fat facets (Faf), a deubiquitinating enzyme, and its substrate Liquid facets (Lqf), an endocytic epsin, promote Delta internalization and Delta signaling in the signaling cells. While Lqf is necessary for three different Notch/Delta signaling events at the morphogenetic furrow, Faf is essential only for one: Delta signaling by photoreceptor precluster cells, which prevents recruitment of ectopic neurons. In addition, the ubiquitin-ligase Neuralized (Neur), which ubiquitinates Delta, is shown to function in the signaling cells with Faf and Lqf. The results presented bolster one model for Neur function in which Neur enhances Delta signaling by stimulating Delta internalization in the signaling cells. It is proposed that Faf plays a role similar to that of Neur in the Delta signaling cells. By deubiquitinating Lqf, which enhances the efficiency of Delta internalization, Faf stimulates Delta signaling (Overstreet, 2004).
Cells with decreased lqf+ activity accumulate Delta on apical membranes and fail to signal to neighboring cells. Three Notch/Delta signaling events were examined in the eye: proneural enhancement, lateral inhibition and R-cell restriction. Loss of lqf+-dependent endocytosis during all three events has identical consequences to loss of Delta function in the signaling cells. It is concluded that lqf+-dependent endocytosis of Delta is required for signaling, supporting the notion that endocytosis in the signaling cells activates Notch in the receiving cells. However, Lqf is not required absolutely for all Delta internalization in the eye. Even in lqf-null cells, which are incapable of Delta signaling, some vesicular Delta is present. Perhaps not all of the vesicular Delta present in wild-type discs results from signaling (Overstreet, 2004).
Genetic studies in Drosophila indicate clearly that deubiquitination of Lqf by Faf activates Lqf activity. Moreover, genetic and biochemical evidence in Drosophila suggests that Faf prevents proteasomal degradation of Lqf. In vertebrates, however, it is thought that epsin is mono-ubiquitinated to modulate its activity rather than poly-ubiquitinated to target it for degradation. If Lqf regulation by ubiquitin also occurs this way in the Drosophila eye, the removal of mono-ubiquitin from Lqf by Faf would activate Lqf activity (Overstreet, 2004).
Whatever the precise mechanism, given that both Faf and Lqf are expressed ubiquitously in the eye, two related questions arise. First, why is Lqf ubiquitinated at all if Faf simply deubiquitinates it everywhere? One possibility is that Faf is one of many deubiquitinating enzymes that regulate Lqf, and expression of the others is restricted spatially. This could also explain why Faf is required only for R-cell restriction. Another possibility is that Faf activity is itself regulated in a spatial-specific manner in the eye disc. Alternatively, Lqf ubiquitination may be so efficient that Faf is needed to provide a pool of non-ubiquitinated, active Lqf. Similarly, Faf could be part of a subtle mechanism for timing Lqf activation. Second, why is Faf essential only for R-cell restriction? One possibility is that there is a graded requirement for Lqf in the eye disc, such that proneural enhancement requires the least Lqf, lateral inhibition somewhat more and neural inhibitory signaling by R2/3/4/5 the most. The mutant phenotype of homozygotes for the weak allele lqfFDD9 supports this idea, as R-cell restriction is most severely affected. Alternatively, Lqf may be expressed or ubiquitinated with dissimilar efficiencies in different regions of the eye disc. More experiments are needed to understand the precise mechanism by which the Faf/Lqf interaction enhances Delta signaling (Overstreet, 2004).
In neur mutants, Delta accumulates on the membranes of signaling cells and Notch activation in neighboring cells is reduced. These results support a role for Neur in endocytosis of Delta in the signaling cells to achieve Notch activation in the neighboring receiving cells, rather than in downregulation of Delta in the receiving cells. Because neur shows strong genetic interactions with lqf and both function in R-cells, Neur and Lqf might work together to stimulate Delta endocytosis. Lqf has ubiquitin interaction motifs (UIMs) that bind ubiquitin. One explanation for how Neur and Faf/Lqf could function together is that Lqf facilitates Delta endocytosis by binding to Delta after its ubiquitination by Neur. This is anattractive model that will stimulate further experiments (Overstreet, 2004).
One exciting observation is that the endocytic adapter Lqf may be essential specifically for Delta internalization. Although, hedgehog, decapentaplegic and wingless signaling pathways have not been examined directly, they appear to be functioning in the absence of Lqf. These three signaling pathways regulate movement of the morphogenetic furrow and are thought to require endocytosis. The furrow moves through lqf-null clones and at the same pace as the surrounding wild-type cells. Moreover, all mutant phenotypes of lqf-null clones can be accounted for by loss of Delta function. Further experiments will clarify whether this apparent specificity means that Lqf functions only in internalization of Delta, or if the process of Delta endocytosis is particularly sensitive to the levels of Lqf (Overstreet, 2004).
Lqf expands the small repertoire of endocytic proteins that are known targets for regulation of cell signaling. In addition to Lqf, the endocytic proteins Numb and Eps15 (EGFR phosphorylated substrate 15) are objects of regulation. In vertebrates, asymmetrical distribution into daughter cells of the alpha-adaptin binding protein Numb may be achieved through ubiquitination of Numb by the ubiquitin-ligase LNX (Ligand of Numb-protein X) and subsequent Numb degradation. In addition, in vertebrate cells, Eps15 is phosphorylated and recruited to the membrane in response to EGFR activation and is required for ligand-induced EGFR internalization. Given that endocytosis is so widely used in cell signaling, endocytic proteins are likely to provide an abundance of targets for its regulation (Overstreet, 2004).
In screens for mutations affecting wing pattern, six alleles of a single complementation group were obtained that cause phenotypes similar to those caused by the loss of Notch signaling, namely severe wing notching, wing vein thickening and bristle tufts. All six alleles fail to complement existing alleles of liquid facets (lqf), and are associated with nonsense or missense mutations in the lqf-coding sequence (Overstreet, 2003). One new allele, lqf1227, truncates the coding sequence after amino acid 119 in the middle of the ENTH domain, the most N-terminal conserved domain, and abolishes Lqf protein expression in vivo. This allele is referred to as lqf-, and it was used for all experiments described in this study. A transgene containing the intact lqf gene (Cadavid, 2000) rescues the lethality of lqf- homozygotes, as well as all of the mutant phenotypes associated with lqf- clones. Lqf encodes the sole ortholog of vertebrate Epsin1 (Cadavid, 2000); a second Drosophila protein, sometimes referred to as Dm Epsin2 (Overstreet, 2003), lacks several conserved domains found in Lqf and vertebrate Epsin1, and appears instead to be the Drosophila ortholog of vertebrate EpsinR (see Mills, 2003), a functionally distinct Epsin-related protein (Wang, 2004).
In imaginal wing discs, signaling by the DSL ligands Delta (Dl) and Serrate (Ser) specifies the wing margin at the dorsoventral (DV) compartment boundary, and can be assayed by boundary-specific expression of wing margin genes (or their protein products), such as cut, wingless (wg) and vestigial (vg). lqf- clones resemble Dl- Ser- clones or N- clones in that they cause the loss of cut, wg and vg boundary-specific expression when they abut or cross the DV compartment boundary, corroborating the Notch-related phenotypes of lqf- clones observed in the adult wing (Wang, 2004).
The loss of margin gene expression in lqf- clones is not cell autonomous. Instead, wild-type cells can rescue the expression of margin specific genes in adjacent lqf- cells (e.g., cut). Similarly, non-autonomous rescue of lqf- clones was observed in the adult, where the presence of wild-type cells can rescue the ability of neighboring lqf- cells to form single bristles. In both respects, as well as in others, lqf- clones resemble Dl- Ser- clones, but differ from N- clones, which show a strictly cell-autonomous loss of Notch target gene expression (Wang, 2004).
Collectively, these data establish an obligate role for Lqf in Notch signaling, and implicate Lqf in sending, rather than receiving, DSL signals (Wang, 2004).
To determine whether Lqf is required in signal-sending cells, the MARCM technique was used to generate lqf- clones that express either Dl or Ser under Gal4 control. Notch is normally expressed in both the D and V compartments of the wing primordium, but is modified in D cells by the action of the glycosyltransferase Fringe (Fng) so that it responds preferentially to Dl signaling from V cells. Ser is expressed predominantly in D compartment cells, and signals in the opposite direction, activating unmodified Notch in V cells. Clones of cells that express Dl under Gal4 control activate Notch strongly in adjacent wild-type cells only when located in the D compartment, as monitored by the expression of margin-specific genes like cut. Conversely, Ser-expressing clones activate Notch strongly only when located in the V compartment. In both cases, the levels of exogenous Dl and Ser expression are several fold higher than the peak levels of endogenous Dl and Ser generated along the DV boundary, and this overexpression autonomously inhibits the activation of Notch in cells within the clones (Wang, 2004).
Clones of lqf- cells that overexpress either Dl or Ser fail to induce margin gene expression, irrespective of where they are located within the wing primordium. Indeed they behave like simple lqf- clones in blocking normal margin gene expression when they abut, or cross, the DV compartment boundary. Thus, Lqf is required in DSL signal-sending cells to activate Notch in adjacent, signal-receiving cells (Wang, 2004).
Intact Dl and Ser normally accumulate in intracellular puncta, some of which co-localize with the endosomal marker Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), as well as at the apical cell surface. By contrast, C-terminally truncated forms of Dl that lack the intracellular domain (DlDeltaC) accumulate predominantly at the cell surface and, like C-terminally truncated forms of Ser (SerDeltaC), cannot activate Notch on the surface of neighboring cells. If such truncated DSL ligands fail to signal because they cannot be endocytosed, replacement of the missing Dl cytosolic domain with heterologous domains that contain other internalization signals should rescue both endocytosis and signaling activity. Moreover, mutations in the internalization signals of these domains should eliminate their rescuing activity. These predictions have been tested and confirmed with two heterologous domains, each containing a different internalization signal (Wang, 2004).
(1) The missing intracellular domain of DlDeltaC was replaced with a 21 amino acid peptide from the Low Density Lipoprotein (LDL) receptor that contains either the wild-type internalization signal FDNPVY, or a mutant signal, ADAAVA. The LDL peptide contains two Lysines; these were replaced by Arginine to avoid their serving as possible acceptors for ubiquitination. Both the wild-type (DlLDL+) and mutant (DlLDLm) chimeric proteins were labeled by the insertion of six copies of the Myc epitope tag in the juxtamembrane portion of the extracellular domain. When expressed in the wing disc, DlLDL+ shows a similar subcellular distribution to wild-type Dl, accumulating on both the apical cell surface and in intracellular puncta. DlLDL+-expressing clones, like wild-type Dl-expressing clones, can induce cut activity in surrounding cells, indicating that the chimeric protein has signaling activity. However, they differ from wild-type Dl-expressing clones in that they only induce cut when located close to the DV boundary. Hence, it is inferred that DlLDL+-expressing clones have reduced signaling activity relative to wild-type Dl-expressing clones, and require the additional boost provided by endogenous signaling from neighboring wild-type cells to activate cut (Wang, 2004).
By contrast, DlLDLm accumulates predominantly on the apical cell surface, but not in intracellular puncta, and lacks signaling activity. Indeed, clones of cells overexpressing DlLDLm that abut the DV boundary block normal Notch signaling across the boundary, as would be expected if DlLDLm can inhibit Notch transduction within the same cell (like wild-type Dl), but is devoid of the capacity to activate Notch in adjacent cells (Wang, 2004).
(2) It was found serendipitously that replacement of the missing cytosolic domain of DlDeltaC with a random peptide, R+, of 50 amino acids (DlR+) also restored normal behavior. DlR+ accumulates in intracellular puncta as well as on the apical cell surface; in addition it activates Notch in neighboring cells. The R+ peptide contains two Lysines that might potentially serve as acceptors for ubiquitination. Replacement of both Lysines with Arginine blocked the rescuing activity of the R+ peptide. The mutant protein, DlRm, accumulated predominantly only on the cell surface and lacked signaling activity; moreover, clones of DlRm that abutted the DV boundary interrupted signaling across the boundary (Wang, 2004).
To assess the possibility that mono-ubiquitination of native Dl, as well as the DlR+ chimera, might suffice to provide an internalization signal, the missing cytosolic domain of DlDeltaC was replaced with Ubiquitin itself, and with a corresponding mutant form of Ubiquitin in which the Isoleucine at position 44 was mutated to Alanine, which functionally inactivates the internalization signal. All seven Lysine residues in each Ubiquitin domain were also replaced by Arginine to avoid additional ubiquitination. Both the resulting proteins, DlUbi+ and DlUbim, accumulated on the cell surface, as well as in intracellular puncta. However, far fewer puncta were found in DlUbim-expressing cells than in DlUbi+-expressing cells, and only DlUbi+ was able to signal to neighboring cells. These data indicate that mono-ubiquitination is sufficient for Dl endocytosis and signalling, and suggest that at least one of the Lysines in the R+ peptide serves as Ubiquitin acceptor, allowing the protein to be internalized and to signal. It is noted that the DlUbi+ protein appears to have only weak signaling activity relative to Dl or DlR+, since induction could only be detected of vg boundary-specific expression, but not cut or wg expression (Wang, 2004).
It is concluded that: (1) the cytosolic domain of Dl is essential for its endocytosis; (2) mono-ubiquitination is sufficient for Dl internalization; and (3) Dl endocytosis is essential for signaling activity (Wang, 2004).
Given that Epsin has been implicated in endocytosis, lqf- cells may fail to send DSL signals because they are generally impaired for endocytosis. However, Dpp, Hh and Wg signaling, Wg internalization, and cell growth and proliferation are not adversely affected by the absence of Lqf, suggesting that endocytosis is not significantly impaired overall (Wang, 2004).
Alternatively, Lqf might be required specifically for the endocytosis of DSL ligands. To assess this possibility, the effects of lqf- clones were first examined in the developing retina, where they have been reported to cause abnormally high levels of Dl on the cell surface, consistent with impaired Dl endocytosis (Overstreet, 2003). Such clones do indeed cause elevated surface expression of Dl, but it was also observed that endogenous Dl transcription (as assayed using a Dl-lacZ reporter gene), is strongly upregulated in the mutant cells, apparently as a consequence of the lack of Notch signaling. Furthermore, Dl staining can be detected in intracellular puncta in such lqf- eye disc clones. Thus, the elevated surface accumulation of Dl observed in lqf- eye clones can be ascribed to elevated Dl expression in the mutant cells, and may not reflect impaired Dl endocytosis (Wang, 2004).
lqf- clones were generated in wing discs expressing uniformly high levels of exogenous Dl under Gal4 control, and Dl staining was compared in lqf- cells and their wild-type neighbors. Under this condition, the level of Dl expression does not vary between wild-type and lqf- cells, simplifying analysis. No difference was detected in the subcellular distribution of Dl between lqf- and adjacent wild-type cells. In both cases, Dl was localized predominantly at the cell surface, as well as in similar numbers of intracellular puncta, many of which co-localize with the endosomal protein Hrs. The same result was obtained in separate experiments in which only the subcellular distribution of endogenous Dl was assayed (i.e., in the absence of overexpressed Dl) (Wang, 2004).
It was reasoned that if the Dl-positive puncta in lqf- clones are indeed endocytic, the appearance of such puncta should change in the absence of hrs activity, which interferes with the maturation of early into late endosomes, and causes the formation of abnormal endosomal structures. To test this, both hrs- and hrs- lqf- clones were generated. Endogenous Dl was found to accumulate in abnormally large puncta in both types of clones, and similar results were obtained when these clones expressed exogenous Dl under Gal4 control. The block in endosomal maturation caused by the removal of Hrs does not interfere with signaling by Dl; nor does it alter the requirement for Lqf. Clones of hrs- cells that express exogenous Dl induce Cut expression in surrounding cells, whereas corresponding hrs- lqf- clones do not (Wang, 2004).
To determine unequivocally whether the abnormal puncta that accumulate Dl in hrs- and hrs- lqf- cells are indeed endosomal, use was made of the finding that Wg secreted from prospective wing margin cells accumulates in similar, abnormally large puncta in hrs- cells positioned at a distance from the secreting cells. The same result was obtained in double mutant hrs- wg- cells, establishing that the accumulation of Wg in these puncta serves as an in vivo marker for endocytosis. Then Wg and Dl staining was examined in triple mutant hrs- wg- lqf- clones that express an HRP-tagged form of Dl under Gal4 control. In this case, as in corresponding hrs- wg- double mutant clones, co-localization of Wg and Dl was observed in large intracellular puncta. Thus, bulk endocytosis of both endogenous and overexpressed Dl appear normal in lqf- cells (Wang, 2004).
Although bulk Dl endocytosis appears unaffected by the absence of Lqf, blockage of a relatively small, but specific, subset of Dl endocytic events might escape detection, and this subset might be crucial for signaling activity. To examine this possibility, Dl was co-expressed together with the E3 Ubiquitin Ligase Neuralized (Neur), under Gal4 control, to drive efficient ubiquitination and internalization of the exogenous Dl. It was reasoned that under these conditions, even modest reductions in the rate of Dl endocytosis might cause an abnormal persistence of Dl at the apical cell surface (Wang, 2004).
Wing discs that express uniformly high levels of Dl under Gal4 control accumulate high levels of Dl on the apical cell surface. However, in discs that co-express high levels of both Dl and Neur, this surface accumulation is strongly reduced and Dl accumulates instead in an abnormally large number of intracellular puncta. Clones of lqf- cells generated in such co-expressing discs do not appear to alter the number or general appearance of these Dl-positive puncta, many of which co-localize with Hrs. However, they do affect the level of Dl staining associated with the apical cell surface (as visualized in discs processed either with, or without, detergent). Such lqf- clones show residual surface staining of Dl, in contrast to neighboring wild-type cells where surface-associated staining is depleted. It is inferred that lqf- cells cannot endocytose Dl as efficiently as their wild-type neighbors, accounting for why a difference was detected under sensitized conditions in which the rate of surface clearance appears to be limiting (Wang, 2004).
Significantly, the residual staining of Dl on the surface of lqf- cells that overexpress Neur and Dl correlates with the failure of these cells to signal. Clones of lqf- cells that overexpress Neur and Dl fail to activate cut in neighboring cells, even though clones of otherwise wild-type cells that overexpress Neur and Dl show enhanced Dl signaling. Hence, it appears that the impairment in Dl endoctyosis detected in lqf- clones in this sensitized background correlates with an absolute block in signaling activity (Wang, 2004).
The cytosolic domain of DSL ligands contains multiple Lysines at least some of which serve as acceptors for Ubiquitin. Lqf contains two Ubiquitin Interacting Motifs (UIMs) (Hofmann, 2001). Hence, mono-ubiquitination of DSL ligands might allow Lqf to target DSL ligands for a special subset of endocytic events that are required for signaling activity. By contrast, bulk endocytosis of DSL ligands mediated by interactions with other Ubiquitin-binding adaptor proteins might not suffice to confer signaling activity. To test this hypothesis, whether the signaling activity of the DlR+ protein depends on Lqf activity, was investigated (Wang, 2004).
Endocytosis and signaling activity of DlR+ depends on the presence of at least one of the two Lysines in the R+ peptide comprising the cytosolic domain. Clones of lqf- cells that express DlR+ fail to induce cut expression in adjacent wing disc cells. However, DlR+ protein in these lqf- clones accumulates both on the apical surface and in intracellular puncta. Moreover, no difference was detected in the punctate, cytosolic accumulation of DlR+ between lqf- and wild-type cells in wing discs that generally overexpress DlR+. Both results indicate that bulk endocytosis of DlR+ is not significantly altered in the absence of Lqf. Because substitution of both Lysines by Arginine blocks internalization and signaling activity of DlRm, it is inferred that DlR+ is targeted for internalization solely by ubiquitination at one or both of these Lysines. Hence, it is suggested that other Ubiquitin-interacting proteins aside from Lqf can target mono-ubiquitinated cargo proteins, such as DlR+ or endogenous Dl, for internalization. However, only Lqf appears able to direct endocytosis of these proteins in a way that allows DSL ligands to signal (Wang, 2004).
Both endocytosis and signaling activity of DlLDL+ depends on the FDNPVY internalization signal. However, unlike either native Dl or DlR+, it was found that clones of lqf- cells expressing DlLDL+ can induce cut expression in adjacent wild-type cells, indicating that the presence of the LDL internalization signal in the chimeric DlLDL+ protein bypasses the requirement for Lqf. As observed for clones of wild-type cells overexpressing DlLDL+, the 'rescued' lqf- clones induced cut only when located close to the DV boundary. Nevertheless, their ability to signal, albeit weakly, contrasts with that of lqf- clones that overexpress native Dl, native Dl plus Neur, or DlR+, all of which are devoid of signaling activity. Hence, it is concluded that the FDNPVY signal directs internalization of DlLDL+ in a manner that permits the protein to acquire signaling activity even in the absence of Lqf activity (Wang, 2004).
Lqf-dependent endocytosis of DSL ligands might be accompanied by modifications of these ligands, either as a pre-requisite for, or a consequence of, signaling activity. To examine this possibility, it was asked whether the size of Dl protein changes as a consequence of Lqf-dependent endocytosis. Initially, clones of wild-type and lqf- cells were generated that express Dl tagged by the insertion of six copies of the Myc epitope in the extracellular juxtamembrane domain, and the profile of Dl peptides that retain the Myc epitope was examined by Western blotting. Under these conditions, similar, complex profiles were observed of Myc-tagged Dl peptides from both wild-type and lqf- cells, corresponding to full-length Myc-Dl protein, as well as several lower molecular weight peptides (Wang, 2004).
This experiment was then repeated using wild-type and lqf- cells that overexpress Neur and Myc-tagged Dl, the sensitized condition under which residual surface expression can be detected of Myc-tagged Dl in lqf-, but not in wild-type, cells. In this case, the profile of Myc-tagged Dl is remarkably simple. Wild-type cells show two bands, one corresponding by size to full-length Myc-tagged Dl (~105 kDa) and the other to a Myc-tagged cleavage product of ~50 kDa. By contrast, lqf- cells show only a single band, corresponding to full-length Myc-tagged Dl. Thus, the failure to clear Dl from the cell surface of lqf- cells is associated with an apparent failure in Dl processing. These results provide evidence for a Lqf-dependent cleavage of Dl that correlates with Lqf-dependent endocytosis and signaling activity (Wang, 2004).
It is noted that the expected size of the Myc-tagged extracellular domain of Dl is ~75 kDa, whereas that of the complementary, Myc-tagged portion of the ligand containing the transmembrane and cytosolic domains is ~40 kDa. Hence, the 50 kDa Myc-tagged cleavage product must be composed of a C-terminal portion of the extracellular domain, and possibly some or all of the transmembrane and cytosolic domains as well. The relationship of this truncated peptide to the active ligand is presently unknown. It could comprise part, or all, of the active ligand, or alternatively, a non-signaling C-terminal fragment cleaved off in the process of generating an N-terminal signaling fragment. Alternatively, it might be a degradation product generated as a consequence of the activation of Notch by Dl (Wang, 2004).
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date revised: 25 August 2008
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